RIG-I Inhibits Hepatitis E Virus Replication by Simultaneously Activating JAK-STAT and NFKB Pathways

POSTER PRESENTATIONS with absolute and relative number of circulating neutrophils and hematic levels of myeloperoxidase (MPO), an enzyme stored in neutrophilic granules and released upon activation. In our experiment ex vivo performed by us, neutrophils released MPO after stimulation with recombinant HMGB1, demonstrating a direct effect of this protein on their activation and granule exocytosis. Finally, upon co-culture with graded doses of neutrophils preactivated or not with HMGB1, T cells were dose-dependently inhibited in their proliferation and expression of activation markers. Conclusions: Our data for the first time highlight a possible protective role of HMGB1 and neutrophils in the early phases of the inflammatory processes involved in NAFLD. Likewise, this initial appreciation of a virtuous interplay between supposedly harmful molecules and myeloid cells warrants further mechanistic studies, aimed at providing exploitable tools for targeted therapy in steatotic liver diseases. FRI-239 INTERFERON REGULATORY FACTOR 1 RESTRICTS HEPATITIS E VIRUS REPLICATION BY ACTIVATING STAT1 TO INDUCE ANTIVIRAL INTERFERON-STIMULATED GENES L. Xu1, X. Zhou1, W. Wang1, Y. Wang1, Y. Yin1, L.J.W. Van Der Laan2, D. Sprengers1, H.J. Metselaar1, M.P. Peppelenbosch1, Q. Pan1. 1 Department of Gastroenterology and Hepatology; 2Department of Surgery, ERASMUS MC, Rotterdam, Netherlands E-mail: [email protected] Background and Aims: Interferon signaling plays an essential role in defending viral infection by the stimulation of hundreds of interferon-stimulated genes (ISGs). Interferon regulatory factor 1 (IRF1) is one of the most important ISGs in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract with HEV infection is largely obscure. This study aims to investigate the action of IRF1 on HEV replication and the interaction with the current off-label treatment, including interferon-α (IFNα) and ribavirin. Methods: Multiple human cell lines including hepatoma cells (Huh7), primary-like hepatic cells (HepaRG), lung epithelial carcinoma cells (A549) and fetal lung fibroblast cells (MRC-5) were used to harbor two HEV models: a subgenomic replicon coupled with a luciferase reporter and a full-length genome can produce infectious HEV particles. Results: In Huh7-based HEV model, over-expression of IRF1 inhibited HEV replication level to 62.5 ± 3% (72 h, mean ± SEM, n = 12, p < .0001) in subgenomic model and 54.8 ± 4% (48 h, mean ± SEM, n = 11, p < .0001) in full-length infectious model. This effect was confirmed in several other cell lines including HepaRG, A549 and MRC-5. Although the efficacy was comparable to high dose of IFNα treatment (1000 IU/mL), IRF1 did not trigger interferon production. Chip-seq data analysis surprisingly revealed that IRF1 was able to bind to the promoter region of signal transducers and activators of transcription 1 (STAT1), a key element of the interferon signaling. Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevated total and phosphorylated STAT1 proteins. This further activated the transcription of 17 tested downstream antiviral ISGs. Furthermore, pharmacological inhibition of JAK-STAT pathway could totally block the antiviral ability of IRF1. Consistently, RNAi-mediated gene-silencing approaches revealed that antiviral function of IRF1 was dependent on the JAK-STAT cascade. Moreover, the induction of ISGs and anti-HEV effect of IRF1 overlapped with IFNα but were potentiated by ribavirin. Conclusions: We demonstrated that IRF1 effectively inhibited HEV replication through the activation of JAK-STAT pathway and the subsequent transcription of a wide range of antiviral ISGs, but independent of interferon production.

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FRI-240 RIG-I INHIBITS HEPATITIS E VIRUS REPLICATION BY SIMULTANEOUSLY ACTIVATING JAK-STAT AND NFΚB PATHWAYS L. Xu1, W. Wang1, X. Zhou1, Y. Wang1, Y. Yin1, L.J.W. Van Der Laan2, D. Sprengers1, H.J. Metselaar1, M.P. Peppelenbosch1, Q. Pan1. 1 Department of Gastroenterology and Hepatology; 2Department of Surgery, ERASMUS MC, Rotterdam, Netherlands E-mail: [email protected] Background and Aims: Interferon-stimulated genes (ISGs) are thought to be the ultimate antiviral effectors of interferons. Although hundreds of ISGs have been identified, little is known regarding their effects on hepatitis E virus (HEV) infection. This study aims to identify the key antiviral ISGs against HEV. Methods: Lentiviral vectors were used to ectopically over-express human ISGs. Their effects were tested in two human liver cell lines including hepatoma cells (Huh7) and primary-like hepatic cells (HepaRG), which harbor a HEV luciferase reporter replicon or a fulllength infectious model. Results: By individually over-expressing 24 ISGs that are previously shown antiviral against different viruses, we identified RIG-I (also known as DDX58) as one of the most potent anti-HEV ISGs. Overexpression of RIG-I inhibited HEV replication by 56 ± 5% (72 h, mean ± SEM, n = 8, p < .0001) in subgenomic model and 58 ± 4 % (48 h, mean ± SEM, n = 10, p < .0001) in full-length infectious model in Huh7 cells. Consistently, RIG-I could also drastically suppress HEV viral RNA by 64 ± 4% (48 h, mean ± SEM, n = 4, p < .05) in HepaRGbased infectious model. Although RIG-I has been reported inducing the transcription of interferon genes in particular cell types, overexpression of RIG-I in our system did not trigger the expression and production of interferons. Strikingly, RIG-I stimulated the expression and phosphorylation of STAT1, which is a key intracellular mediator of interferon signaling. This subsequently led to activation of JAK-STAT cascade, resulting in the induction of a subset of antiviral ISGs. However, pharmacological inhibition of JAKSTAT pathway only partially blocked the anti-HEV effect of RIG-I. Concurrently, we found that over-expression of RIG-I could also activate the NFκB pathway, inducing important downstream antiviral genes/ chemokines including RANTES and IL-1β. Conclusions: This study identified RIG-I as one of the most potent anti-HEV ISGs. Mechanistically, without triggering interferon production, RIG-I simultaneously activates the JAK-STAT and NFκB pathway to induce antiviral ISGs and chemokines. FRI-241 MIR-155: A DOUBLE FACED MICRORNA WITH RESPECT TO ITS IMPACT ON TUMORIGENESIS IN BOTH NATURAL KILLER CELLS AND THEIR TARGET HEPATOCYTES M.A. Rahmoon1, R.A. Youness1, A.I. Gomaa2, I. Waked2, H.M. El Tayebi3, A.I. Abdelaziz4. 1Pharmaceutical Biology department, German University in Cairo, Cairo; 2Hepatology, National Liver InstituteMenoufiya University, Menoufiya; 3Pharmacology and Toxicology Department, German University in Cairo; 4Biology Department, American University in Cairo, Cairo, Egypt E-mail: [email protected] Background and Aims: miR-155 is among the most prominent oncogenic miRNA in different malignancies. In our previous work, miR-155 was overexpressed in HCC, upregulating IGF-2 and IGF-1R and downregulating IGFBP3 leading to a final outcome of exaggerated carcinogenicity. In immune cells, BIC/miR-155 expression is increased in activated B and T cells. However, its role in Natural Killer cells (NKs) has recently been shed the light on. NKs immune-surveillance is mediated through cellular cytotoxicity and cytokine secretions. miR-155 is among the top miRNAs showing differential expression in resting and activated NKs. However, it has never been probed in NKs of HCC patients. Thus, in diversion from the conventional

Journal of Hepatology 2016 vol. 64 | S425–S630